The Ah receptor (AHR) has been traditionally studied in terms of its ability to mediate transcriptional effects through binding to dioxin-response elements (DRE). The AHR is known to be involved in a wide array of physiological processes, including T cell function and liver vascular development. We have tested the hypothesis that the AHR can regulate gene expression in the absence of binding to its cognate response element using an AHR DNA binding mutant. One class of genes that were observed to be repressed by an AHR-A78D DNA binding mutant were acute phase liver inflammatory genes (e.g. SAA1, CRP). Next, AHR ligands that can mediate acute-phase gene repression activity without inducing DRE-mediated transcriptional activity were identified; these compounds have been termed Selective Ah receptor modulator (SAhRM). One compound, SGA360 is capable of inhibiting cytokine-mediated induction of acute-phase gene expression without inducing a cognate response element (DRE) effect. The mechanism(s) of this SAhRM-induced AHR- dependent anti-inflammatory activity has not been established. Therefore, in this application the first specific aim will determine the precise mechanism of selective AHR ligand-mediated repression of acute-phase response gene expression. This will be accomplished using cell culture models (e.g. Huh7 hepatoma cells). A combination of cell treatment with SAhRMs followed by the use of the following techniques; siRNA, protein blot analysis, chromatin immunoprecipitation assays, promoter analysis, cell-based reporter assays, EMSA and co-immunoprecipitation analysis will be used to determine the protein(s) involved in acute-phase gene induction that is modulated by SGA360-AHR complex. There is also a need to develop additional SAhRMs with higher affinity and potency that will enhance their anti-inflammatory activity, especially in in vivo models. Thus, the second specific aim will use two independent computer modeling alogorithms of ligand binding to the AHR ligand-binding pocket and our knowledge from previous structure-activity studies to guide structure-activity studies, which will lead to the development of high affinity selective ligands that exhibit anti-inflammatory properties. The aim will utilize computer modeling and ligand docking programs, organic synthesis of new compounds, cell-based acute-phase gene repression assays, EMSA, ligand competition assays and skin and liver inflammation assays in mice to identify structural modifications that enhance SGA360-mediated repression of acute-phase gene expression. These studies taken together will allow future testing on their therapeutic potential in appropriate chronic inflammatory disease animal models (e.g. cancer, Crohn's disease). PUBLIC HEALTH RELEVANCE: Activation of the Ah receptor (AHR) has been demonstrated to repress cytokine-mediated acute phase gene expression in liver. Selective AHR ligands that can mediate acute phase gene repression without eliciting a dioxin response element-driven transcriptional response have been identified. These studies will explore the mechanism of selective AHR ligand activity, which may lead to the development of the AHR as a therapeutic target to treat chronic inflammatory diseases. Also more potent selective AHR ligands will be developed.